Radio- and fluorescence-labelled Cetuximab for multimodal imaging

Objectives:

Radio (⁶⁴Cu⁺⁺) and fluorescence (Alexa Fluor 488) labelled Cetuximab (C225) offers the possibility of multimodal imaging of epidermal growth factor receptor (EGFR). ([⁶⁴Cu]Cu-NOTA)₇-C225-(Alexa Fluor 488)⁷ was synthesized and the imaging and biodistribution has been proved by small animal PET and ex vivo studies of cryo-sections of xenotransplanted mice.
Methods:
1) Synthesis of (NOTA)_x-C225-(Alexa Fluor 488)_y (C225-Conjugate). The C225 solution (Erbitux, Merck KGaA, Germany) was buffer-exchanged with a NaHCO3 solution (50 mM, pH 6.0) containing NaCl (150 mM) using a Jumbosep 30 k molecular weight cut-off concentrator (Pall Life Sciences, Germany). NOTA (2-(pisothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, p-SCN-Bn-NOTA, Macrocyclics, USA) and Alexa Fluor 488 (Alexa Fluor 488 carboxylic acid 2,3,5,6-tetrafluorophenyl ester, Invitrogen, United Kingdom) has been conjugated to C225 lysine amino residues in a single step. Just prior to protein conjugation, NOTA and Alexa Fluor 488 were dissolved in HEPES-buffer (400 μL, 500 mM, pH 7.2) and added to the C225 solution to achieve a molar reaction ratio of 10:1 (NOTA:C225) and gently manually agitated. The reaction mixture was allowed to incubate in the dark at 25°C for 24 h without further stirring. Excess NOTA chelator and dye were removed by passage of the reaction solution through a Jumbosep. The buffer-exchange was performed with an NH4OAc solution (50 mM, pH 6.0) containing NaCl (150 mM). Protein concentration and the number of conjugated dye molecules per mAb were determined by UV/VIS absorption at 280 nm and 495 nm (excitation maxima of Alexa Fluor 488), respectively. The number of NOTA introduced was determined using MALDI-TOFMS. 2) Radiolabeling with ⁶⁴Cu. A solution of [⁶⁴Cu]CuCl₂ in HCl (0.04 M, 50 MBq) was added to the C225-conjugate (100 μg) plus MES buffer (100 μL, 200 mM, pH 6.1) in 2 mL protein low-binding tubes (Eppendorf, Germany), vortexed immediately and incubated at 30°C for 30 min. Radiochemical yield (RCY) was determined by radio-ITLC. Before ITLC-analyses, free TETA chelator (32 μL; 0.1 μg/μL) was added to the reaction mixture for quenching the labelling reaction and fixing unreacted radionuclide. Therefore a second incubation at 30°C for 30 min was necessary. As stationary ITLC-phase, a 10 cm ITLC-SA strip (Varian GmbH, Germany) and as mobile phase a 0.9% NaCl solution has been used. The radio labelled antibody was purified by spin filtration using Microcon 30 k (Millipore). 3) Biodistribution. Squamous cell carcinoma (FaDu) tumour bearing NMRI nu/nu mice were studied with PET at 24 h p.i. The fine distribution of the probe was then characterized using whole body cryosectioning. The ⁶⁴Cu-activity was compared with the fluorescence distribution.
Results:
The BFC NOTA and Alexa Fluor 488 were successfully simultaneously conjugated to C225. The resulting conjugate is (NOTA)₁-C225-(Alexa Fluor 488)₇. Radionuclide labelling was achieved within 30 min with optimization of the molar ratio of NOTA:C225: Alexa Fluor 488 during the conjugation. The radioimmunoconjugate exhibited high accumulation in xenotransplanted FaDu tumours in mice after 24 hours. The ⁶⁴ Cu-activity concentration correlated well with the fluorescence in most tissues except the liver.
Conclusions:
Dual-labelled immunoconjugates represent a potential probe for translational application in tumour detection, evaluation of long-term distribution and stability studies of the labelled antibody.
Research support: This project was partially supported by “Kompetenzverbund Strahlenforschung” (grant 02NUK006A-E).